Methods and kits for detection of thromboxane a2 metabolites

ABSTRACT

Methods, compositions and kits are provided for measuring aspirin&#39;s anti-thrombotic effectiveness on a subject. Included are a novel assay for quickly and specifically measuring TxA2 metabolite levels in urine and correlating the levels with aspirin dose in a subject. The methods, compositions and kits utilize a novel anti TxA2 metabolite antibody.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application No. 60/748,788 entitled “METHODS,COMPOSITIONS AND ASSAYS FOR DETECTION OF THROMBOXANE A2 METABOLITES INBIOLOGIC FLUID” filed Dec. 9, 2005, and to U.S. Provisional PatentApplication No. 60/793,428 entitled “ANTIBODIES TO THROMBOXANE A2METABOLITES, AND USES THEREOF filed Apr. 19, 2006, the disclosure ofboth are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

a. Field of the Invention

The invention generally relates to antibodies that bind two or morethromboxane A2 metabolites, and more particularly to antibodies thatbind common epitopes in two or more target thromboxane B2 metabolitesderived from thromboxane A2 and to the uses for these preparedantibodies.

b. Background Art

Aspirin (acetyl salicylic acid) has been in use in the United States forat least 100 years, with approximately 80 million aspirin tablets beingconsumed in America each day (self medicated or physician prescribed).Beyond pain relief, aspirin has been shown to reduce risk ofcerebrovascular ischemia, myocardial infarction, angina, and recurrentblockage of arteries (collectively referred to as thrombotic disease(Antithrombotic Trialists' Collaboration, BMJ, 324:71-86, 2002)).Thrombotic disease is one of the world's leading causes of morbidity andmortality, and its prevalence is increasing (Lopez et al., Nat. Med.,4:1241-1243, 1998).

The Food and Drug Administration has recently recommended that personssuffering from thrombotic disease take from between 50 mg aspirin/day to325 mg aspirin/day. However, higher doses of aspirin pose health risksin certain segments of the population, including stomach irritation,ringing in ears, allergic reactions and in children, Reye's syndrome.

Aspirin has been shown to inactivate cyclooxygenase (also known as COXor prostaglandin G/H synthase), a membrane bound enzyme responsible forthe oxidation of arachidonic acid to prostaglandin G₂ (Awtry et al,Circulation, 101: 1206-1218, 2000). This reaction is a precursor to theformation of a variety of prostanoids, including thromboxane A2 (TxA2),a potent platelet aggregator, and metabolites of thromboxane A2 such asthromboxane B2. In general, aspirin has been shown to reduce COXactivity (there are actually two COX enzymes termed COX-I and COX-II)and thereby reduce the levels of downstream prostanoid development.Inactivation of these pathways ultimately limits thrombotic events bysuppressing at least the ability of platelets to aggregate (Hamberg etal., PNAS, 72:2994-2998, 1975; Ellis et al., Science, 193:1135-1137,1976).

Although the mechanism of aspirin inhibition of COX-1 is understood,recent studies suggest that not all users respond to aspirin to the samedegree. A lack of response to aspirin in a user is generally referred toas “aspirin resistance.” Person's suffering from aspirin resistancetypically show either a lack of biochemical changes while on aspirin,i.e., user shows no or little reduction in TxA2 (and TxB2), plateletactivation and/or aggregation, or the user may experience an ischemicevent while on aspirin (Bhatt et al., Nature Rev., 2:15-28, 2003; Hankeyet al., Lancet, 367:606-617). In either case, the prevalence of aspirinresistance in the population has been reported to be between 5 and 57%(Gum et al., Am J Cardiol, 88:230-235, 2001; Tarjan et al., Orv. Hetil,140:2339-2343, 1999; Sane et al., Am J Cardiol, 90:893-895, 2002;Helgason et al., Stroke, 25:2331-2336, 1994). Identification of whetheror not an individual is aspirin resistant prior to or during athrombotic event would be extremely useful to a healthcare professional.An aspirin resistant individual would obviously receive an alternatedosage, drug or modified anticoagulant therapy.

Typically, studies using aspirin have focused on optimizing the amountof aspirin required for an individual to reduce the risk associated withthrombotic disease. Blood based assays have been developed that measurein vitro platelet aggregation as a measure of aspirin's effectiveness.However, these methods are not quantitative and can be affected byfactors that are unrelated to aspirin sensitivity. In addition, aquantitative immunoassay is available for 11-dehydrothromboxane B2 (aTxA2 metabolite) detection in urine requiring the use of a polyclonalantibody to 11-dehydrothromboxane B2. The assay is helpful in thataspirin effectiveness can be determined from a subject's urine, but thepolyclonal antibody does not provide highly reproducible or specificresults.

U.S. Pat. No. 6,967,083 (herein '083) to Ens provides methods foridentifying an optimal minimal aspirin dose for a patient that isspecifically tailored to the patient's platelet response levels. Themethod utilizes a solid substrate coated by an agent capable of reactingwith 11-dehydro thromboxane B2 in the urine, but provides little or noguidance as to how to reproducibly achieve this detection result giventhe tools described in the patent. For example, no agents in the '083patent are described or shown that react with 11-dehydro thromboxane B2or other thromboxane A2 metabolites.

As such, there is a need in the art to develop a highly specific andreproducible assay that measures effectiveness of aspirin in reducingplatelet aggregation in a subject. The assay should be effective atmeasuring a dose of aspirin tailored to a particular subject forreducing thrombotic disease. In addition, there is a need in the art fora highly specific and reproducible assay that quickly identifies aspirinresistant individuals, thereby facilitating a health care professionalsdetermination on a best course of treatment in the absence of aspirin.

Against this backdrop the present invention has been developed.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for measuring the anti-thromboticeffectiveness of aspirin treatment on a subject. The methods alsoinclude identifying subjects that display aspirin resistance ortolerance. In some embodiments these methods take less than five hoursand more typically less than three hours to complete. Embodiments of theinvention typically include correlation between a subject's urine TxA2metabolite level and the aspirin dose administered to the subject. Inparticular embodiments the invention includes correlation between asubject's urine 11dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2metabolite levels and the aspirin dose administered to the subject.

The present invention provides immunoassay-based kits for measuring TxA2metabolite levels in urine. Kits provide assays that can be completedwithin five hours and more typically three hours. Kits also include amonoclonal antibody having reactivity with both 11 dh TxB2 and11-dehydro-2,3 dinor thromboxane B2.

Finally, the present invention provides novel tools for identifying TxA2metabolites in a biologic fluid, e.g., urine. In a preferred embodiment,novel tools are provided capable of identifying two or more TxA2metabolites. In a particularly preferred embodiment, the tool comprisesa monoclonal antibody derived from hybridoma number cells describedherein. The monoclonal antibody having reactivity with two or more TxA2metabolites and more particularly with the TxA2 metabolites: 11 dh TxB2and 11-dehydro-2,3 dinor thromboxane B2.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Data within the following Figures is often expressed in terms of 11 dhTxB2, for purposes of the Figure legends and Figures, 11 dh TxB2includes 11 dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2.

FIG. 1 is a linearity plot showing actual values of samples at variousdilutions.

FIG. 2 is a recovery assay plot showing the excellent correlationbetween observed and expected values when testing samples usingembodiments of the present invention.

FIG. 3 is a bar graph showing the high kit stability of embodiments ofthe present invention.

FIG. 4 is a box plot comparison of 11 dh TxB2 against samples fromaspirin users.

FIG. 5 is a frequency plot of urinary 11 dh TxB2 values from subjects onaspirin or subjects not on aspirin.

FIG. 6 a is a standard curve of OD readings at 405 nm for varied 11dhTxB2 concentrations (between 100 pg/mL and 1,000 pg/mL) and FIG. 6 bis a standard curve of OD readings at 405 nm for varied 11dhTxB2 and11-dehydro-2,3 dinor thromboxane B2.

FIG. 7 is a histogram representing 11dhTxB2 levels in 55 normalindividuals, the 11dhTxB2 determined in accordance with embodiments ofthe present invention.

FIG. 8 shows a comparison of 11dhTxB2 levels in normal individuals (noaspirin) and individuals treated with either 81 mg, 162 mg or 325 mgaspirin.

FIG. 9 shows average 11dhTxB2 levels in normal, non-aspirin, users, andusers who have been treated with 81 mg, 162 mg or 325 mg aspirin.

FIG. 10 is a bar graph showing Pg 11dhTxB2/mg creatinine in CADpatients, three doses of ASA were used (81, 162, 325 mg).

FIG. 11 is a bar graph showing pg 11dhTxB2/mg urine creatinine in femaleobese patients with high cardiac risk undergoing general surgicalprocedures, with or without aspirin.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention include methods and assays formeasuring the effectiveness of aspirin treatment in a subject, includingmethods and assays for identifying aspirin resistant subjects within apopulation. The methods and assays of the invention partly rely onimproved immunoassays for highly sensitive, specific, timely and highlyreproducible assays targeted at detection of one or more TxA2metabolites in bodily fluids and especially in urine. Note that bodilyfluids may also include blood plasma.

In one embodiment, the present invention provides a highly sensitiveurine-based monoclonal ELISA for detection of 11 dhTxB2 and, in apreferred embodiment, at least one additional TxA2 metabolite. In aparticularly preferred embodiment, the invention provides a highlysensitive urine-based monoclonal ELISA for detection of both 11 dhTxB2and 11-dehydro-2,3 dinor thromboxane B2 (11 dh2,3dTxB2). As demonstratedherein, the detection of multiple TxA2 metabolites provides additionalsensitivity and reproducibility as compared to assays that detect onlyone metabolite, i.e., 11 dhTxB2.

Anti 11 dhTB2 and 11 dh2,3dTxB2 Monoclonal Antibody

The present invention provides antibodies that specifically recognizetwo or more TxA2 metabolites and in particular recognize 11 dhTxB2 and11 dh2,3dTxB2. As used herein the term antibody refers to proteins andfragments thereof, such as Fab, F(ab)₂, and Fv, where the fragments arecapable of recognizing and binding either 11 dhTxB2 and/or 11dh2,3dTxB2.

The present invention provides monoclonal antibodies identified fortheir capacity to selectively bind the epitope presented by thestructure shown in formula 1.

The specificity is defined by the fact that antibodies of the presentinvention react with 11 dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2but remain unreactive with 2,3 dinor TxB2, PGD2, TxB2, 6-keto PGF1a,PGE2, PGEM or PGFM. (See, for example, Formulas 2-5 and Example 6).

Formulas 2-5, respectively:

In accordance with the present invention, monoclonal antibodies areselectively produced by immunizing an animal, e.g., mouse, rat, human,rabbit, horse, goat, etc., with an antigen having an epitope in commonwith 11 dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2 (see, forexample, Formula 1). The method includes immunizing an animal; selectingcells from the immunized animal that has been activated to expressantibodies against two or more TxA2 metabolites, such as an epitope incommon with 11 dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2; fusingthe harvested cells to myeloma cells e.g., SP 210, NS10, (or other likecells) to form hybridoma cells; selecting the hybridoma cells thatsecrete antibodies reactive to the appropriate antigen; andcharacterizing the antibody against the epitope in Formula 1.

The antibodies of the present invention can be isolated and purified bymethods well known in the art, such as, for example, ammonium sulfateprecipitation.

Various assays can be used to determine an antibody's immunogenicreactivity, such as competitive immunoprecipitation assays and the like.

Hybridoma cells that produce antibodies of the invention can be selectedand isolated by various known methods in the art. In some embodiments,myeloma cells and lymphocytes activated against two or more TxA2metabolites are cultured together in selection medium capable of killingthe unfused myeloma cells but not the lymphocytes. Hybridoma cell linesof the invention form when myeloma cells fuse with lymphocytes capableof producing antibodies against two or more TxA2 metabolites. Fusedcells are capable of survival in the selection medium while both unfusedmyeloma and lymphocytes are eliminated with time.

The potency or affinity that antibodies of the present invention havefor two or more TxA2 metabolites is determined by enzyme-basedcompetition assays (see Examples below) and by its utility in reactingwith TxA2 metabolites in a subject's urine. For example, an antibody ofthe present invention shows at least 50% cross-reactivity with two ormore TxA2 metabolites and preferably 50% cross-reactivity with 11 dhTxB2 and 11-dehydro-2,3 dinor thromboxane B2. In some embodiments, thecross-reactivity of the antibody is at least 50% with one of the TxA2metabolites and at least 75% with an alternative TxA2 metabolite.

Embodiments of the present invention provide a significant improvementover previously developed polyclonal antibodies that recognize only 11dhTxB2. As shown in Table 1 below, polyclonal antibodies against 11dhTxB2 have proven to have suspect reproducibility toward binding of the11 dhTxB2 epitope, especially as compared to the monoclonal antibodiesof the present invention. In addition, whereas the polyclonal antibodiesfound in the prior art bind one TxA2 metabolite, 11 dhTxB2, themonoclonal antibodies of the present invention have been shown toreproducibly interact and bind at least two of the TxA2 metabolites, 11dhTxB2 and 11 dh2,3dTxB2. As such, the monoclonal antibodies of thepresent invention provide for enhanced specificity in diagnostic testingtoward TxA2 metabolites and further for use in identifying aspirinresistant subjects.

In one embodiment, the antibodies of the present invention are obtainedusing the open form of 11 dhTxB2 as the immunogen or immunizing antigen.In some embodiments, especially due to its size, the 11 dhTxB2 isconjugated to a carrier protein, e.g., bovine serum albumin, goat serumalbumin, etc. The coupled immunogen-carrier is then used to immunize amouse or other like animal.

The production of the antibody can be performed by methods known in theart, for example, I. Lefkovits, Ed., (1996) Immunology Methods ManualAcademic Press, Inc., San Diego, Calif. (incorporated herein byreference in its entirety). In typical embodiments the antibody is amonoclonal antibody. Hybridoma cell lines that produce the monoclonalantibodies of the present invention are typically produced by a fusionof an immortalized cell line with a B-lymphocyte and cells selected forantibody production that have affinity against 11 dhTxB2 and preferablyboth 11 dhTxB2 and 11 dh2,3dTxB2. Clones were selected for sensitivityand selectivity toward both 11 dhTxB2 and 11 dh2,3dTxB2.

Hence embodiments of the invention include any monoclonal antibody thathas affinity toward and binds to at least one TxA2 metabolite. Inpreferred embodiments, the invention includes any monoclonal antibodythat has affinity toward and binds to more than one TxA2 metabolite, andin more preferred embodiments the TxA2 metabolites are 11 dhTxB2 and 11dh2,3dTxB2. In addition, embodiments of the present invention includecell lines that produce these antibodies, and in particular, hybridomacell lines.

Urinary 11dhTxB2 Competitive ELISA Assay

An important part of antiplatelet therapy in cardiovascular disease isaspirin, known for its antiplatelet effects. Low dose aspirin blocksmore than 95% of platelet COX-1 activity and reduces cardiovascularevents by as much as 25% in patients with arterial vascular disease.Unfortunately, aspirin is not effective in all individuals. Thisphenomenon has been deemed aspirin resistance, and 5-57% of aspirinusers fall within this category of not responding to the antiplatelettherapy of aspirin.

Measurements of stable metabolites to TxA2, such as 11 dhTxB2 and 11dh2,3DTxB2, which are found in urine, provide an excellent measure ofTxA2 production by platelets and thus an improved tool for analyzingaspirin's effectiveness.

The present invention provides a urinary 11dhTxB2 Competitive ELISAAssay. An illustrative standard curve was prepared (see FIG. 6) byspiking known amounts of 11 dhTxB2 into low level urine diluted 1:5 inTris assay buffer (0.1M Tris, 0.1% BSA) or into assay buffer alone. 100μl of standards or actual samples (having taken known amounts of aspirinand diluted 1:5 in Tris assay buffer) was added to wells coated withgoat anti-mouse antibody, followed by the addition of 50 μl of 11dhTxB2-AP (alkaline phosphatase) tracer and 50 μl of 11 dhTxB2 mAb. Thereactions were then allowed to incubate for a predetermined amount oftime, typically with shaking at room temperature. In preferredembodiments the incubation is about two hours in length. Each well isthen washed with a wash buffer and approximately 200 μl of substrateadded (p-nitrophenyl-phosphate, pNPP). The reactions are then incubatedagain for approximately thirty minutes. A stop solution is added to eachwell, and the plate read for OD at 405 nm.

Results from the assay provide for the amount of 11dhTxB2 and 11dh2,3dTxB2 present in the sample (note that results are compared to thestandard curve). The amount of the two metabolites present in the sampleis indirectly proportional to the optical density read at 405 nm. Forclinical samples, creatinine values are also obtained, and data fromassays presented as pg metabolite/mg creatinine.

In some cases where the assay reliability is known or where the accuracyof the assay is not critical, a standard curve can be avoided.

Polyclonal Antibody (Prior Art) vs Monoclonal Antibody of the Invention

A monoclonal-based assay of the invention was compared to a standardpolyclonal assay using twelve urine controls, run multiple times over aperiod of six months. The controls were derived from aspirin users aswell as non-users. The % CV (coefficient of variance) range in themonoclonal assay was 0.3%-12.9% with an average of 3.7% (see Table 1).The % CV range in the polyclonal assay was 0-35.8% with an average of9.9%. In comparing standard curves, the R² (coefficient of regression)value of the monoclonal assay averaged 0.9973 compared to 0.9877 for thepolyclonal assay. To further compare the mAb and pAb assays, anadditional urine panel was established consisting of ten samples fromboth aspirin users and non-aspirin users, and metabolites from the twoassays compared. The correlation (r) between the methods was 0.944,confirming the clinical relevance of the monoclonal-based assay of theinvention. Thus, this sensitive, urine-based monoclonal ELISA for TxA2metabolites detection eliminates the potential for analytical variablesthat can occur in other assays.

Note that precision data collected on four different samples (n=69)showed good intra-assay precision (average of 1.7%) as well as goodinter-assay precision (also 1.7%). TABLE 1 Monoclonal Antibody andPolyclonal Antibody Comparison Polyclonal Antibody Antibody of theInvention Sample No. Mean (pg/mL) % CV Mean (pg/mL) % CV 1 184 14.0 5044.4 2 210 7.5 756 3.5 3 323 8.9 1069 5.2 4 158 12.6 493 5.2 5 279 6.11136 2.1 6 822 15.6 3303 2.4 7 166 11.6 453 2.8 8 272 12.2 1176 3.4 9794 6.7 2860 5.3 10 188 9.5 439 6.1 11 305 6.6 1115 2.0 12 762 7.5 30882.3 Mean % CV 9.9 3.7

Data shown in Table 1 provide strong evidence that the mAb based ELISA,recognizing two TxA2 metabolites, provides a significant improvement(reproducibility and specificity) in measurement of TxA2 metabolites ascompared to polyclonal based detection systems. As such, the assays ofthe present invention provide a significant benefit in clinical use whenanalyzing aspirin effectiveness on subjects.

The following examples are offered to further illustrate the inventionand are not meant to limit the invention in any manner.

EXAMPLES

Example 1

Preparation of 11-dehydro Thromboxane-hapten Carrier Immunogen

11-dehydro Thromboxane B2 was purchased from Cayman Chemical, CatalogNumber 419504. The identity of the compound can be verified by massspectroscopy (Finnegan ICQ, negative ion mode) and by HPLC. The compoundwas purified to greater than 98% purity using normal phase silicic acidchromatography. Three milligrams of the purified compound was thensuspended in 100 μl of absolute ethanol. The ethanolic solution of11-dehydro Thromboxane B2 was then added to 500 μl of 0.1M potassiumphosphate buffer, pH of 7.4, and 5 mg Keyhole Limpet Hemocyanin (CapeCod Associates), vortexed, and transferred to a 5 ml V-bottom vialequipped with a magnetic stirrer and charged with 5 mg of solid EDC. TheV-bottom vial was wrapped in aluminum foil and placed on a magnetic stirplate at ambient temperature (˜22° C.) overnight. The contents of thevial were then dialyzed through a 10,000 MW cut-off membrane for 8 hoursagainst 7.4 liters of 0.1M potassium phosphate buffer, pH 7.4. Thevolume was standardized to 5 ml with distilled water and 100 μl aliquotswere frozen at −20° C. for immunization (aliquots termed immunogenherein).

Example 2 Preparation of Tracers Used in Antibody Screening

Anhydrous dimethyl formamide (DMF) was prepared by distillation andstorage over molecular sieves. Dry DMF was used to prepare 10 mMsolutions of N-hydroxy succinimide, dicyclohexyldicarbodiimide, and11-dehydro Thromboxane B2. Each compound was prepared in a 10 mlreactivial that was oven dried and stored in a desiccator. In a new, dry5 ml V-bottom reactivial, 10 μl of each solution was passed, vortexedbriefly, sealed with a septum cap and allowed to incubate at ambienttemperature overnight. The next day, 250 μl of 0.1M borate buffer, pH8.5, was added, along with 2,000 Units of alkaline phosphatase. Theresulting mixture was incubated in the dark for approximately 30 minutesat ambient temperature, then purified over a 30×2 Sephadex G-25 mediumcolumn. Fractions were eluted using 0.1M Tris buffer, pH 7.4. Onemilliliter fractions were collected and pooled based on the fractions inwhich activity to p-nitrophenyl phosphate was detected. The tracer wasdiluted to a specific activity of 0.5 AU/μl/s, and 50 μl of tracersolution was used to screen specific antibody in 96-well microplatescoated with goat anti-mouse IgG.

Example 3 Immunization of BALB/C Mice

Four to six week-old BALB/C mice (purchased from Charles River) wereinjected into the peritoneum with equal volumes of immunogen (˜100 μg)and Freunds complete adjuvant. A second substantially identicalimmunization was given each mouse 14 days later and a blood testobtained on day 24, i.e., blood test given to each mouse 10 days afterthe second immunization. A number of mice showed good antibody bindingresponse to 11-dehydro Thromboxane B2 tracer. Mice showing a high titerof serum antibody to 11-dehydro Thromboxane B2 were given a boost of 100μg immunogen in complete Freunds adjuvant and a second test bleed wastaken on day 44. Those mice with continued high titers were given asecond booster injection of immunogen in incomplete Freunds adjuvant.Five days after the second boost, target mice were ready for removal ofspleen.

Example 4 Preparation, Characterization and Purification of HybridomaAntibodies

A fusion reagent was prepared by first weighing out 4.2 g ofpolyethylene glycol (PEG) 4000. The PEG was then liquefied byautoclaving. While the PEG is still liquefied, 1.5 ml of DMSO (q.s. to10 ml with DPBS and CaCl₂ (anhydrous) (50 mg/500 ml) and MgCl₂(hexahydrate (50 mg/500 ml)) is added. The solution can be stored at 4°C.

To prepare for the fusion procedure, the fusion reagent (42% PEG4000+15% DMSO in DPBS with Ca and Mg) is warmed to 37° C. Approximately50 ml sterile RPMI 1640 is added to a 50 ml conical tube and placed at4° C.

Approximately 60 ml of HAT selection medium (20% HI FBS+1X HAT+1XL-glutamine+1X pen/strep+10% NCTC-109 medium+3 ml hybridoma cloningfactor (IL-6 secreted from cell line which is purchased from Fisher)) isadded to RPMI 1640. The HAT selection medium is filtered through a 0.2μm filter unit.

Myeloma cells (SP2/0 cells) were prepared by obtaining 18-20×10⁶ cellswhich were spun down at 1000 rpm for 7 to 8 minutes. The supernatant wasaspirated and the cell pellet re-suspended in 10 ml cold RPMI 1640. Thecells were washed a second time and re-suspended in 5 ml cold RPMI 1640.Myeloma cells were recounted to ensure cell numbers.

Target mice were euthanitized using CO₂. The left side of the mouse wassprayed with 70% ethanol and the spleen removed using sterile scissorsand forceps. The removed spleen was placed into a sterile P60 dish withapproximately 5 ml RPMI 1640. The spleen was rinsed and transferred intoa second sterile P60 dish. The spleen was perfused and then minced withsterile forceps to remove the connective tissue. The minced spleen wasthen transferred into a sterile 15 ml conical tube and the clumps oftissue allowed to settle to the bottom of the tube for two minutes. Thecells were then agitated using a pipet and transferred (without theclumps) to a new 15 ml conical tube. The cells were spun down at 1000rpm for 7 to 8 minutes. Supernatant was removed and the cellsre-suspended in 3 ml of cold RPMI 1640. One milliliter of the splenocytesuspension was transferred into a second 15 ml sterile conical tube. Thecells were pelleted as above and approximately one milliliter of heatinactivated FBS with 5% DMSO was added to the cells and the combinationfrozen. These cells were saved in case the fusion procedure failed.

Eight milliliters of cold RPMI 1640 was added to the 2 ml of remainingsplenocytes. The cells were then spun down at 1000 rpm for 7 to 8minutes. The pelleted cells were re-suspended in 5 ml of cold RPMI 1640and counted (use multiple dilutions).

With regard to the fusion, a 4:1 ratio of splenocytes:myeloma cells wascombined into a 50 ml conical tube. The combined cells were pelleted at1000 rpm for 7 to 8 minutes and the supernatant aspirated. The cellpellet was loosened by agitating the tube with a finger. Over the courseof one minute, 1.5 ml of fusion reagent was added to the pellet and themixture agitated for 20 to 30 seconds. Over the course of the nextminute, 10 ml of warm RPMI 1640 was added to the cells. The cells werethen spun at 1000 rpm for 7 to 8 minutes. The supernatant was removedfrom the cell pellet and the cells re-suspended in 50 ml of HATselection medium. Cells were plated into 8 plates and allowed toincubate in a 37° C. incubator (5% CO₂) for 4 to 5 days.

Unfused cells were found dead after the 4 to 5 day period. New mediumwas combined into the plates including additional HAT selection medium.The cells were allowed to grow for 10 to 14 days.

Example 5 Hybridoma Screening Assay

Fusion cell supernatants were screened using a goat anti-mouse coatedplate purchased from Cayman Chemical (Catalog number 400009).Approximately 100 μl of supernatant from each plate was added to theCayman plate along with 100 μl of 11-dehydro ThromboxaneB2-Acetylcholinesterase tracer (see above). Note that appropriatepositive and negative controls were run on the Cayman plate, i.e., 100μl HAT selection medium, 100 μl diluted mouse serum. Reactions wereallowed to incubate overnight at ambient temperature. Plates were washedwith appropriate wash buffer and 300 μl/well of Ellman's reagent buffer(substrate) added. Those cells corresponding to supernatant that had astrong absorbance at 415 nm were expanded.

In particular, cells from individual well that tested positive wereexpanded into 6-well plates in HAT medium. Once cell growth in thesevessels provided sufficient testing material, supernatants werere-screened to eliminate false positives and to eliminate clones thatstopped producing appropriate antibody. Positive clones were expendedinto T25 flasks to provide sufficient material to further characterizeantibodies of the present invention.

Example 6 Preparation of A Monoclonal Antibody That Recognizes Two orMore Thromboxane A2 Metabolites

Two forms of 11dhTxB2 are in an equilibrium (open and closed). Asanalyzed by HPLC, the compound in ethanol was found to be predominantlyin a closed form (93%), the remaining 7% being in an open form. Afterevaporation of the solvent, the carboxyl group of 11dhTxB2 (200 μg) wascoupled to an amino group of bovine serum albumin (2 mg) by theN-succinimidyl ester method in the presence of phosphate buffer, pH 7.4.(Hosoda, H, et al., Chem. Pharm. Bull. 29:1969-1974, 1981). Theconjugate thus prepared was stored in the same buffer. Three femaleBALB/c mice 6 weeks of age were first immunized by intraperitonealinjection of the conjugate (5.5 μg of 11dhTxB2, 80 μg of protein), whichwas emulsified with an equal volume of complete Freund's adjuvant. Twomore injections were given every 2 weeks in the same way except thatincomplete Freund's adjuvant was used. The last immunogen was given in0.2 ml of saline without adjuvant. The antibody was titrated using11-[³H]dhTxB2 methyl ester, which was dissolved in phosphate buffer (pH7.4) and therefore in an open form. Three days after the last antigeninjection, the spleen was removed from a mouse with the highest titer ofantibody. Fusion of the spleen cells with an aminopterin-sensitivemyeloma cell line (SP2/0-Ag14) was performed using 50% polyethyleneglycol-1000 by the method of Goding (Goding, J. W., J. Immunol. Methods39:285-308, 1980). After a 12-day incubation in 96-well culture plates,the medium containing hypoxanthine, aminopterin, and thymidine wasreplaced by a medium containing hypoxanthine and thymidine in whichUltroser G substituted for serum. After a 2-day culture in thisserum-free condition, an aliquot of the medium was removed for titrationof antibody by radioimmunoassay using 11-[³H]dhTxB2 methyl ester.Selected hybridoma cells were cloned twice in soft agar according to themethod of Kennet (Kennet, R. H., in Monoclonal Antibodies (Kennet, R. H.and McKearn, T. J., Eds.), pp.372-373, 1980). A clone producinganti-11dhTxB2 antibody was injected into the peritoneal cavity of BALB/cmice previously treated with pristine. The antibody in the ascites fluidwas collected with ammonium sulfate at 50% saturation, and was furtherpurified by the use of a protein A-Sepharose column (Oi, V. T andHerzenberg, L. A., in Selected Methods in Cellular Immunology (Mishell,B. B. and Shiigi, S. M., Eds.), pp. 351-372, 1980). About 20 mg of IgGwere obtained from one mouse (Hayashi Y, et al., Anal. Biochem. 187:151-159, 1990).

Example 7 Monoclonal Antibody Specificity

Antibody clones of the invention were tested to determinecross-reactivity against various Thromboxane A2 metabolites and otherlike compounds. Table 2 provides percent cross reactivity of antibodiesof the present invention against these various compounds: TABLE 2 CrossReactivity Compound % Reactivity 11 dh TxB2 100% 11-dehydro-2,3 dinorthromboxane B2 166% 2,3 dinor TxB2 1.93% PGD2 0.2% TxB2 0.05% 6-ketoPGF1a <0.01% PGE2 <0.01% 2,3,4,5-tetranor-20-Carboxy 13,14- <0.01%dihydro-15-keto PGE2 (PGEM) 2,3,4,5-tetranor-20-Carboxy 13,14- <0.01%dihydro-15-keto PGE2α (PGFM)

Data in Example 7 shows the high reactivity of monoclonal antibodies ofthe present invention toward 11 dh TxB2 and 11-dehydro-2,3 dinorthromboxane B2. Specificity is illustrated by the antibodies failure toreact with the other enumerated compounds.

Example 8 Kits of the Invention Show a High Level of Precision

Kits of the invention were tested for precision between three differentplates/kit lots. Three urine samples were chosen of low, moderate, andhigh TxA2 metabolite values (L, M, and H). Each sample was run on 24wells/plate over three plates from three separate kit production lots.Values were obtained and the mean, standard deviation, and %coefficients of variation (% CV) were calculated and shown for eachsample for inter- and intra-assay precision. TABLE 3 Precision TestingInter-Assay Mean Intra- Sample No. Mean Std Dev. Precision AssayPrecision H 3380 334 9.9% 5.7% M 1399 116 8.3% 6.7% L 424 63 14.9% 10.3%

Example 9 Kits of the Invention are Highly Accurate and Sensitive

Linearity of the kits of the present invention were monitored bymeasurement of TxA2 metabolites in the urine of subjects, where theurine was diluted to three different dilutions. Five different samples(Urine 1-5) at four different dilutions each (1:2, 1:5, 1:10 and 1:20)were run using the kits of the invention. Values are shown in Table 4.Dilution factor, mean, standard deviation, and % CV are shown for eachsample. The data illustrates the utility of the kits of the invention.TABLE 4 Linearity of Kits of the Invention Dilution Urine 1 Urine 2Urine 3 Urine 4 Urine 5 1:2 (0.5) 2867 2395 2837 3115 3640 1:5 (0.2)2734 2377 2717 3190 3824 1:10 (0.1) 2670 2340 2708 3187 4049 1:20 (0.05)2618 2372 2509 3105 3912 Mean 2722 2371 2693 3149 3856 SD 108 23 136 46171 % CV 4.0% 1.0% 5.0% 1.4% 4.4%

The data in Table 4 was plotted on a linearity plot as shown in FIG. 1.The data in Table 4 and FIG. 1 show the excellent sensitivity andaccuracy of the kits of the present invention.

A recovery assay plot was performed using kits of the invention. A urinesample containing a high value of TxA2 metabolites was serially diluted1:1.25 and run on a kit of the invention. A value was obtained forDilution #1, and the expected concentrations of each dilution calculatedbased on a 1.25-fold dilution of the observed value of Dilution #1. Theobserved values were compared to the expected value and are shown inFIG. 2. The data of FIG. 2 illustrates the highly accurate and sensitivenature of the kits and methods of the invention.

It was further determined that kits of the invention are highly stableover long periods of time. As shown in FIG. 3, three control sampleswere tested using the kits of the invention, each sample being testedover a 12 month period. The data in FIG. 3 shows that the methods andkits of the invention are useful for testing samples over an extendedperiod of time.

Example 10 Clinical Findings for 1,500 pg TxA2 Metabolites/mg CreatinineThreshold

Initially, 171 urine samples were taken from subjects either off aspirinor on 81 or 325 mg aspirin/day. TxA2 metabolite amounts were determinedfor each sample using the methods and kits of the present invention.Values were normalized for urinary dilution by dividing TxA2 metaboliteamounts by creatinine concentrations.

FIG. 4 shows a box plot comparison of 11 dh TxB2 baseline levels(representing all TxA2 metabolite amounts) against each category ofaspirin user (none, 81 mg or 325 mg). Boxes in FIG. 4 represent 75/25percentiles and the horizontal line within the box represents the medianfor each group. Dots are samples outside the 90/10 percentile bars.

354 urine samples from subjects either on or off aspirin therapy wererun using the methods and kits of the present invention. Values werecalculated and normalized for urinary dilution by dividing by creatinineconcentration, and the frequency of each concentration was calculatedand shown in a frequency plot, FIG. 5. Subjects on aspirin are plottedwith solid bars and subjects not on aspirin are plotted using hatchedbars.

Aspirin response data was then plotted for the 354 samples graphically,as shown in Table 5. Positive samples are below 1500 pg/mg creatininecutoff, whereas negative samples are those that are above the 1500 pg/mgcreatinine cutoff.

The data shows the utility of using a 1500 pg TxA2 metabolite/mgcreatinine cutoff. TABLE 5 Clinical Response Aspirin Ingestion ResultsPresent Absent Positive (<1500 pg/creatinine) 251 9 Negative (>1500pg/creatinine) 16 78

Example 11 Clinical Findings Using a Urinary TxA2 Metabolite BasedCompetitive ELISA Assay

As shown in FIGS. 4-5, clinical data can be obtained using theabove-described methods. Standard curves (FIG. 6) showed goodreproducibility, with % CV in the range of 0.3-3.9% (6 a) and detectsboth 11 dh TxB2 and 11-dehydro-2,3 dinor thromboxane B2 (see FIG. 6 b).The assay detection range was suitable for measurement of 11dhTxB2 in apopulation (62-3122 pg 11dhTxB2/mg creatinine—see FIG. 7). The healthysamples in the study were normally distributed with an average value of1119 pg/mg, and aspirin use resulted in decreased patient values (81,162 and 325 mg/day values at 418, 471 and 377 pg/mg, respectively) (seeFIGS. 8 and 9). All three levels of aspirin doses are statisticallysignificant when compared to the healthy, no aspirin controls.

Note that there was more variability in the 81 mg and 162 mg samplesthan in the 325 mg samples, as these dosages may not be optimal for somepatients in reducing 11dhTxB2 levels. There were samples in the aspirintreated groups with high levels of 11dhTxB2, which may representaspirin-resistant individuals.

For purposes of FIGS. 7-9, Table 3 provides the population that astested: TABLE 3 Methods - Population Population A, n = 46 Healthy, noprevious cardiac events, no aspirin Population B Healthy, no previouscardiac events, ± aspirin no aspirin, n = 9 81 mg aspirin, n = 5 325 mgaspirin, n = 31 Population C Patients hospitalized with previous cardiacevents, + aspirin 81 mg aspirin, n = 42 162 mg aspirin, n = 33 325 mgaspirin, n = 31Other assays can also be used in conjunction with the mAb.

Example 11 Clinical Findings in Patients with Coronary Artery Disease orObesity Using a Urinary 11dhTxB2 Based Competitive ELISA Assay

Two patient groups were evaluated for ASA response using 11 dhTxB2/mgurine creatinine in urine. A first group consisted of fifty patientshaving coronary artery disease (CAD) (herein group A) and the secondgroup consisting of twenty obese female patients having elevated cardiacrisk (herein group B). Note that group B patients presented for generalsurgical procedures. Group A was evaluated peri-procedurally on threedifferent doses of ASA (81 mg) first day and then at day thirty and daysixty when administered 162 and 325 mg ASA respectively. Levels of 11dhTxB2 are increased in patients that are ASA resistant. Group Bpatients were screened for ASA response pre-procedure.

The data for each group is shown in FIGS. 10 (group A) and 11 (group B).The data shows that 11 dh TxB2 is a readily available marker that canquantify insufficient inhibition of TxA2 production by platelets. Thetest may be suitable for pre-procedural screening to determine theeffective dose of ASA on an individual patient basis. The present datashows that TxA2 metabolite testing in urine has great potential toassist clinical decisions as alternatives to anti-platelet therapies.

It is understood for purposes of this disclosure that various changesand modifications may be made to the invention that are well within thescope of the invention. Numerous other changes may be made which willreadily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein.

The list of citations to patents, patent applications and/orpublications cited herein are each hereby incorporated by reference forall purposes:

1. A method for identifying aspirin resistance in a subject comprising: administering to the subject a first dose of aspirin; collecting a urine sample from the subject; determining an amount of two or more thromboxane A₂ metabolites in the urine sample; normalizing the amount of the two or more thromboxane A₂ metabolites to an amount of a standard protein in the subject's urine to obtain a first thromboxane A₂ metabolite/standard protein ratio; and comparing the first thromboxane A₂ metabolite/standard protein ratio to a threshold ratio value wherein a thromboxane A₂ metabolite /standard protein value below the threshold value indicates an absence of aspirin resistance in the subject.
 2. The method of claim 1 further comprising: administering to the subject a second dose of aspirin different from the first dose of aspirin; collecting a second urine sample from the subject; determining an amount of the two or more thromboxane A₂ metabolites in the urine sample; normalizing the amount of the two or more thromboxane A₂ metabolites to an amount of a standard protein in the subject's urine to obtain a second thromboxane A₂ metabolite/standard protein ratio; and comparing the first and second thromboxane A₂ metabolite/standard protein ratios; wherein a subject having substantially the same first and second thromboxane A₂ metabolite/standard protein ratio is identified as being aspirin resistant.
 3. The method of claim 2 wherein the first dose and second dose of aspirin are at least 50 mg apart.
 4. The method of claim 1 wherein the two or more thromboxane A₂ metabolites are at least 11-dehydro Thromboxane B₂ and 11-dehydro-2,3-dinor Thromboxane B₂.
 5. The method of claim 4 wherein the determining amount of thromboxane A₂ metabolites comprises a monoclonal antibody capable of binding to 11-dehydro thromboxane B₂ and 11-dehydro-2,3-dinor Thromboxane B₂.
 6. The method of claim 5 wherein the monoclonal antibody recognizes an epitope common to both 11-dehydro Thromboxane B₂ and 11-dehydro-2,3-dinor Thromboxane B₂.
 7. The method of claim 1 wherein the subject is suffering from or suspected of suffering from thrombotic disease.
 8. A kit for detecting thromboxane A₂ metabolites in a urine sample from a subject comprising: a monoclonal antibody capable of binding two or more thromboxane A₂ metabolites; and a signal molecule capable of competing with thromboxane A₂ metabolites in the urine sample for binding to the monoclonal antibody; wherein the two or more thromboxane A₂ metabolites in the subject's urine sample competes with a known amount of signal molecule for binding to the monoclonal antibody thereby indicating the amount of the two or more thromboxane A₂ metabolites in the urine.
 9. The kit of claim 8 further comprising: a substrate modified for capture of the monoclonal antibody and for provision of a constrained space to contact the monoclonal antibody with both the signal molecule and urine sample.
 10. The kit of claim 8 wherein the two or more thromboxane A₂ metabolites are at least 11-dehydro Thromboxane B₂ and 11-dehydro-2,3-dinor thromboxane B₂.
 11. The kit of claim 10 wherein the monoclonal antibody recognizes an epitope common to both 11-dehydro Thromboxane B₂ and 11-dehydro-2,3-dinor Thromboxane B₂.
 12. The kit of claim 11 wherein the monoclonal antibody recognizes the 11-dehydro Thromboxane B₂ metabolite and 11-dehydro-2,3-dinor Thromboxane B₂ metabolite with similar binding ratios, the ratio of 11-dehydro Thromboxane B₂: 11-dehydro-2,3-dinor Thromboxane B₂ is from 0.5:1 to 1:0.5.
 13. The kit of claim 12 wherein the ratio of 11-dehydro Thromboxane B2: 11-dehydro-2,3-dinor Thromboxane B₂ is from 0.6:1 to 1:06.
 14. The kit of claim 12 wherein the substrate is modified with a goat anti-mouse antibody capable of tethering the monoclonal antibody to the substrate.
 15. Use of a monoclonal antibody capable of binding two or more thromboxane A₂ metabolites for determining aspirin resistance in a subject.
 16. The use of a monoclonal antibody of claim 15 wherein the two or more thromboxane A₂ metabolites are at least 11-dehydro Thromboxane B₂ and 11-dehydro-2,3,-dinor Thromboxane B₂.
 17. The use of a monoclonal antibody of claim 16 wherein the monoclonal antibody recognizes the 11-dehydro Thromboxane B₂ metabolite and 11-dehydro-2,3-dinor Thromboxane B₂ metabolite with similar binding ratios, the ratio of 11-dehydro Thromboxane B₂: 11-dehydro-2,3-dinor Thromboxane B₂ is from 0.5:1 to 1:0.5.
 18. The use of a monoclonal antibody of claim 16 wherein the ratio of 11-dehydro Thromboxane B2: 11-dehydro-2,3-dinor Thromboxane B₂ is from 0.6:1 to 1:06. 